DESCRIPTION: Every year, more than 1 million surgical procedures involving the partial excision of bone, bone grafting and fracture repair are performed in the USA, at an estimated cost of more than $5 billion. Well-established clinical approaches are restricted to autograft and allograft transplantation. However, they are limited in access and availability and associated with complications, including graft devitalization and subsequent resorption. As an alternative, tissue engineering using 3D scaffolds may have a huge impact in the future in the repair of bone defects. Developing clinically-relevant bioengineered bone involves challenges in terms of mass transport requirements due to high metabolic activity of bone cells. We have shown that macro/microporous scaffolds produced by a hybrid 3D-bioplotting/porogen-leaching technique can enhance cell ingrowth and produce bone tissue capable of supporting hematopoiesis as well as long term self-renewal of mesenchymal stem cells (MSCs) after implantation in mice. In this work, we are proposing a hybrid 3D-bioplotting/thermally induced phase separation (TIPS) technique. The orthogonally-interconnected channels produced by 3D-bioplotting can provide an ideal environment to guide bone ingrowth. The matrix surrounding these channels will be composed of micropores generated by the TIPS technique, where the pore size and pore morphology can be controlled by manipulating the process parameters and scaffold composition. We have demonstrated the potential of this technique for producing scaffolds made of poly (lactic-co-glycolic acid) (PLGA). Since bone is largely composed of hydroxyapatite (HA), we will perform a design of experiments (DOE) to investigate the effect of HA nanoparticles (nHA) and scaffold architecture on in vitro bone ingrowth and mechanical properties of these scaffolds. This study will test the hypothesis that the hierarchical composite scaffolds will enhance osteogenic differentiation of human MSCs and bone formation through the following specific aims: Aim 1. Develop hierarchical PLGA/nHA constructs as scaffolds for bone tissue engineering. Hypothesis: The hybrid 3D-bioplotting/TIPS will allow designing hierarchical PLGA/nHA scaffolds with the target mechanical properties (modulus > 5 MPa), whereas the DOE will lead to optimal scaffold topologies. Outcome: These scaffolds will offer a highly controlled internal architecture, with orthogonal channels surrounded by a microporous matrix, while the presence of nHA will improve the mechanical properties. Aim 2. Evaluate and monitor the cellular metabolism during in vitro cell growth within the scaffolds. Hypothesis: The new scaffolds will combine the benefits of improved cell migration and oxygen/nutrient transport, while providing a bone-mimicking matrix to enhance cell adhesion (over 50% of the seeded cells) (r) and bone matrix deposition (at least 20% increase compared to CellCeram commercial scaffolds). Approach: Human MSCs will be seeded on the scaffolds and cultured under static and dynamic conditions in media that will induce osteogenic differentiation. Cell attachment, viability, proliferation, and differentiation will be determined for up to 8 weeks based on DNA quantification, alkaline phosphatase (ALP) activity, bone differentiation markers, and microcomputed tomography (uCT).